A laser microscope focuses laser light on a specimen through an objective lens and scans the specimen with the laser light in two directions (X-direction and Y-direction) orthogonal to each other along a plane perpendicular to an optical axis to acquire a planar image of the specimen. On the other hand, a plurality of tomographic images (Z-stack images) along the Z-direction are obtained by changing a distance along the optical axis direction (Z-direction) between the objective lens and the specimen, whereby the laser microscope forms a three-dimensional image of the specimen.
In a confocal laser microscope which is the mainstream in laser microscopes, a light flux of reflected or scattered light or fluorescence generated on a specimen is transmitted by an optical system, and light flux transmitted through a pinhole disposed at an optically conjugated position with respect to a light focusing point on the specimen is received by a detector. Disposing the pinhole makes it possible to filter the light generated on the specimen other than the light focusing point. Therefore, the confocal laser microscope is operable to acquire an image with a good S/N ratio.
Further, a multi-photon microscope increases a photon density on the focal plane by focusing laser light by an objective lens having a large numerical aperture NA. Thereby, a fluorescent molecule absorbs a plurality of photons (N photons) simultaneously and then the fluorescent molecule is excited with energy N times of common energy in intensity. For example, in a two-photon microscope, two photons hit a fluorescent molecule simultaneously and fluorescence having a wavelength half that of common fluorescence is observed. A probability in which two photons hit the fluorescent molecule simultaneously is very small and therefore, no emission occurs from a region other than the focal point. Therefore, even without the pinhole described above, the multi-photon microscope is operable to acquire only specimen information of the focal point.
In observing a biospecimen, the biospecimen is often observed through a cover glass in a state in which the biospecimen is immersed in a broth. Further, generally, the objective lens is designed so that an imaging performance is best at a position immediately below the cover glass having a predetermined thickness and therefore, an aberration due to the objective lens is generated when an optical path length to an observation plane is changed. In observing the inside of a biospecimen, it is necessary to acquire an image at an observation position equivalent to a depth where a broth or biological tissues are transmitted, and an aberration is generated in proportion to a distance from a position immediately below the cover glass to the observation position, resulting in a decrease in resolution.
This aberration will be described in detail with reference to FIG. 2A and FIG. 2B. FIG. 2A and FIG. 2B are diagrams each schematically illustrating an aberration generated depending on a depth of a specimen to be observed. To simplify the description, the objective lens is assumed to be designed to be optimized in observing a medium having a uniform refractive index. FIG. 2A illustrates a light flux 100 in observing a medium having a uniform refractive index used in the design. FIG. 2A illustrates that the light flux 100 is focused on one point without aberration. Contrary to the above, FIG. 2B illustrates a light flux 110 in observing a surface of a specimen at the depth D. The light flux 110 is refracted on an interface 111 between the medium in contact with the objective lens and the specimen and therefore, the light flux 110 is not focused on one point due to the generated aberration.
For example, when the objective lens is a dry lens, a space between the objective lens and the specimen is filled with air. Therefore, a refractive index of the medium (air) between the objective lens and the specimen is 1.0, which is different from a refractive index of a biospecimen (e.g., 1.39). Therefore, an aberration is generated in proportion to a difference between the refractive index of the medium between the objective lens and the specimen and the refractive index of the biospecimen, as well as an observation depth of a biological body. On the other hand, when the objective lens is a water immersion lens, a space between the objective lens and the specimen is filled with water. Therefore, a refractive index of the medium (water) between the objective lens and the specimen is 1.333, which is closer to the refractive index of the biospecimen than the refractive index of air. Therefore, the water immersion lens is suitable for observing a deep portion of a biological body. However, the refractive index of the biospecimen is not equal to the refractive index of water and therefore, an aberration is also generated due to a difference between the refractive index of the biospecimen and the refractive index of water. Therefore, a decrease in resolution is still problematic.
Further, the cover glass also has variations in the thickness thereof within a tolerance range from a design value (e.g., 0.17 mm). An aberration is generated in proportion to a difference of an actual thickness of the cover glass from the design thickness due to a difference between a cover glass refractive index of 1.525 and a biospecimen refractive index of 1.38 to 1.39. A spherical aberration having a phase distribution symmetrical with respect to an optical axis is generated due to these deviations from the design value.
As one means for solving image quality deterioration resulting from the aberrations described above, a correction ring is known. The correction ring is a ring-shaped rotary member provided for an objective lens, and distances between lens groups constituting the objective lens are changed by rotating the correction ring. Thereby, an aberration due to an error in a thickness of the cover glass or in observing a deep portion of a living body is cancelled. A scale is marked on the correction ring and, for example, rough numerical values such as 0, 0.17, and 0.23 are indicated with respect to the thickness of the cover glass. Then, adjusting the scale of the correction ring in accordance with a thickness of an actually used cover glass makes it possible to adjust the distances between the lens groups in such a manner as to optimize the distances in accordance with the thickness of the cover glass (e.g. see Patent Literature 1).
Further, a technique of compensating for a generated aberration by a wave front conversion element which is one example of an aberration correction device is also known. In this technique, a matrix-drivable shape variable mirror element is disposed on an optical path of a microscope, a wave front shape is modulated by the shape variable mirror element based on wave front conversion data measured in advance, and modulated light waves are allowed to be incident on a specimen, whereby an aberration-corrected image having a high imaging performance is acquired (e.g. see Patent Literature 2).
Further, as the wave front conversion element, a spatial light modulation element of an LCOS (Liquid Crystal on Silicon) type is known, in which a voltage is applied to each pixel of a liquid crystal element where pixels are arrayed in a matrix manner and a refractive index of liquid crystal is changed to display a phase distribution which cancels a wave front aberration (e.g. see Patent Literature 3). The spatial light modulation element of an LCOS type is an electro-optic phase modulation element of a reflection type in which a direct liquid crystal layer is formed in an address unit prepared using CMOS technology and a phase modulation amount of each pixel is controlled by a drive voltage.
Further, a microscope control method for controlling an aberration correction amount based on a distance between an objective lens and a specimen using such correction means is also known (e.g. see Patent Literature 4).